Method and device for sonicating a biological sample

ABSTRACT

The present disclosure relates to a device for sonicating a biological sample. In one embodiment, a sample tube holder is pivotally suspended in a mount of a sonication device, thus allowing for a rotational degree of freedom and/or lateral movement that provides an optimized contact area between the sonotrode and the sample tube. Also disclosed is a method for sonicating a biological sample using the device described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the priority and benefit of EuropeanPatent Application No. 19167291.4, filed Apr. 4, 2019, and the priorityand benefit of European Patent Application No. 18165846.9, filed Apr. 5,2018, the contents of which applications are incorporated by referenceherein.

FIELD OF THE INVENTION

The present invention belongs to the field of analytical systems forconducting biological or biochemical assays. Within this field, itrelates to processing of a liquid biological sample, such as a samplecontaining biomolecules, with the aid of ultrasonic treatment.

BACKGROUND

Primary biological samples to be analyzed, for example, in the contextof in-vitro diagnostics, often require pre-analytical treatment beforethey can be processed by a biochemical or biophysical analyzer or thelike.

For instance, in the context of detecting a pathogen, access of theanalytical reagents to a specific analyte within that pathogen may beachieved by a typical preparation step in which viral particles orbacterial cells are lysed such that the respective contents arereleased, before further measures for the enrichment of the analyte inquestion may be applied. Standard lysis procedures for most of thecommon viral particles and bacterial cells are well-stablished and knownto the person of skill in the art.

However, certain pathogens require a more rigorous treatment for asuccessful lysis, including species of the Mycobacterium tuberculosiscomplex (MTBC), in the following also referred to as “mycobacteria”.These bacteria are enveloped by a relatively thick and complex cell wallthat exhibits a considerably higher robustness than their counterpartsfound in most other clinically relevant bacteria.

Samples suspected to contain mycobacteria—or other challengingbiological material—may be pre-treated by the application of ultrasound,also referred to as sonication or ultrasonication. The approach isgenerally known and established in the art. However, in some instances,the efficient transmission of ultrasound from a sonication device to asample can be difficult to achieve. Some approaches in the art rely onsonication while the respective sample vessel is submersed in a liquidmedium, often simply water. In that case, the water actually transportsthe vibration caused by the ultrasonic waves to the sample vessel andthus the sample within, wherein a portion of the primary kinetic energyis usually lost as heat. A more efficient approach involves the directtransfer of ultrasonic energy through physical contact between asonotrode and the respective vessel. Here, as well, the energy transfermay be impeded if the contact area between the sonotrode and the wall ofthe sample vessel is not optimal.

Pertinent approaches applied in the art include, for example, U.S. Pat.No. 6,686,195, disclosing a weight controlling the degree of couplingbetween a sonotrode and a sample vessel. However, the surfaces of samplevessels are usually not ideally even, but exhibit certain material-and/or production-based tolerances that can reduce the effectivephysical coupling and thus the transmission of ultrasonic energy.

The present disclosure describes an approach that avoids suchshortcomings in the art.

SUMMARY

In a first aspect described herein, a device is disclosed for sonicatinga biological sample contained in a sample tube. The device includes atleast a sample tube holder, a sonotrode, and an actuator.

In one embodiment, the sample tube holder is flexibly suspended from apivot engaged to a mount within the sonication device. The pivot allowsfor sample the tube holder to rotate about an x-axis in verticaldirection. As long as the sample tube holder is not rotationallydeflected, it holds the respective sample tube in a vertical orientationalong a z-axis essentially following the direction of gravity and beingperpendicular to the x-axis, such that the tube and the sample withinare held substantially upright. The sample tube holder has an opening toat least one side to allow for physical contact between the tube and thesonotrode. In some embodiments, the mount is configured to permitmovement of the sample tube holder along the x-axis. In otherembodiments, the sample tube holder is configured to permit movement ofthe sample tube within the sample tube holder along the x-axis. Inparticular embodiments, rotation about the x-axis, movement of thesample tube within the sample holder along the x-axis, and/or movementof the sample holder along the x-axis within the pivot combine toprovide an optimal engagement between the sonotrode and the sample tube.

The sonotrode includes a sonication area through which the ultrasound istransmitted to the sample tube upon contacting its essentiallyround-shaped side wall. The sonotrode and the sample holder are alignedsuch that the vertical center of the sonication area—along the z-axis—ison the same height as the rotational pivot point—represented by thex-axis—about which the sample tube holder is rotatable. The sonotrode ismounted on a guiding rail such that it can be moved with the aid of theactuator towards or away from the sample holder following a y-axis. Thelatter is perpendicular to both the x-axis and the z-axis. Forsonication, the sonicating area can be brought into contact with theside wall of the sample tube through the respective opening in thesample tube holder. Guidance of this movement is improved by the guidingrail being fixed at one end to the mount for the sample tube holder. Arotational movement of the sample tube and thus the sample tube holdermay be induced by the contact of the sonicating area by applying apre-defined force to the sample holder via the sonotrode, if due tounevenness of the tube's side wall surface or in case of a tapered orconical tube wall a portion of the tube wall comes into contact with thesonicating area prior to the tube wall's other portions, thus optimizingthe actual contact area and thereby the transmission of ultrasound. Aconcave surface of the sonicating area contributes to maximize thecontact area in a lateral direction, along the x-axis.

As a further aspect, a method for sonicating a biological samplecontained in a sample tube using the device described above is disclosedherein.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1A shows a perspective side view of an embodiment of the disclosedsonication device.

FIG. 1B shows the sonication device of FIG. 1A in a sonication position.

FIG. 2A shows a cross-sectional side view of the sonication device ofFIG. 1.

FIG. 2B shows a cross-sectional side view of the sonication device ofFIG. 1B, which is in a sonication position.

FIG. 3A shows a perspective side view of the sonication device of FIG.1A without the guide rail shown.

FIG. 3B shows a perspective side view of the sonication device of FIG.1B, which is in a sonication position, without the guide rail shown.

FIG. 4A shows a cross-sectional view of the sonication device of FIG.3A.

FIG. 4B shows a cross-sectional view of the sonication device of FIG.3B, which is in a sonication position.

FIG. 5A displays various stages (i-iv) of the movement of the sonotrodetowards a sample tube holder with concomitant adjustment of the sampletubes along the x-axis with increasing force.

FIG. 5B shows the rotatable portion of a sample holder.

FIG. 5C shows a perspective view of sample tubes fully engaged with atransmittal unit of a sonotrode.

DETAILED DESCRIPTION

A first aspect described herein is a sonication device for sonicating abiological sample contained in a sample tube with an essentiallyround-shaped side wall, the sonication device comprising:

-   -   a sample tube holder pivotally suspended in a corresponding        mount, allowing for a rotational movement of the sample tube        holder in vertical direction about an x-axis being a rotational        axis defined by a pivot, wherein the sample tube holder is        configured, in the absence of a rotational movement, to hold the        sample tube in a vertical orientation along a z-axis being        essentially parallel to the direction of gravity and        perpendicular to the x-axis, and wherein the sample tube holder        comprises an opening towards at least one side;    -   a sonotrode comprising a sonicating area for applying ultrasound        to the sample tube, the sonicating area comprising a concave        surface and being mounted at essentially the same height as the        pivot with the x-axis being essentially at the height of the        vertical center of the sonicating area, wherein the sonotrode is        mounted on a guiding rail for moving the sonotrode along a        y-axis perpendicular to the x-axis and the z-axis towards or        away from the sample tube holder, wherein the mount for the        sample tube holder is fixed to one end of the guiding rail with        the opening of the sample tube holder facing the sonicating area        such that the sonicating area in a sonication position is in        physical contact with a side wall of the sample tube in the        sample tube holder;    -   an actuator for moving the sonotrode on the guiding rail along        the y-axis relative to the sample tube holder.

This sonication device enables the skilled person to optimize thetransmission of ultrasonic energy from the sonotrode to the biologicalsample in the respective sample tube and thereby efficiently treat it,such as, for instance, to lyse robust pathogens like mycobacteria andrelease their biochemical contents such as nucleic acids.

The interaction of the pivotally suspended sample tube holder with theforce-controlled sonotrode allows for a geometrical flexibility,compensating for variations in or on the surface of the sample tube sidewall.

In case of unevenness of the tube wall, which may, for instance, be owedto tolerances in the production process of the tube, the contact areabetween the sonicating area may be reduced as previously mentioned, suchthat the energy transfer to the tube and thus the biological sampletherein may be hampered. In the sonication device described herein, thesample tube which is, in the absence of a rotational movement of thesample tube holder, held essentially vertically along the z-axis, mayhave a slightly uneven or warped surface. Alternatively or additionally,the tube may in some embodiments have a tapered shape towards its topor, more often, its bottom. For instance, the tube may in someembodiments have a conical shape.

In such or similar cases, the side wall of the sample tube may not forman ideal plane surface. More precisely, along the vertical dimension ofthe sample tube wall to be contacted with the sonicating area, certainportions of the surface may protrude in relation to other portions. Inthe case of a rigidly mounted sample tube holder, the sonicating areawould be pressed against the tube wall upon moving the sonotrode to thesample tube holder without the possibility to adapt location ororientation. In the case of lesser applied force, the sonicating areawould touch the tube wall only at its protruding portion(s), resultingin a non-optimal contact area and the related loss of energytransmission. In the case of higher applied force, on the other hand,the sonotrode may deform the tube, which may endanger the integrity ofthe sample tube. The latter case may even lead to loss of the samplematerial, including contamination of the sonication device and posingsecurity risks to the operating personnel in case the biological sampleindeed contains pathogens.

However, the sonication device described herein avoids these risks byproviding geometrical flexibility for optimizing the contact areabetween the side wall of the sample tube and the sonicating area. Moreprecisely, in the above-described case, a sonotrode being pressedagainst the side wall with a pre-defined force would not cause thesample tube holder to remain in the conformation leading to a diminishedcontact area, but the sample tube holder could rotate around the x-axisdefined by the pivot point of the pivot upon contact of the tube wallwith the sonication area of the sonotrode. For instance, if the tube hasa tapered shape towards its bottom and is held vertically within thesample tube holder, then a vertical, substantially even, sonicating areawould usually first contact the upper portion of the tube wall. Uponapplication of a pre-defined force to the tube wall from the actuatorvia the sonotrode, the tube can now initiate a rotational movement inwhich its contacted upper portion follows the movement of the sonotrode,while the lower—so far not contacted—portion is rotated towards thesonicating area opposite to the movement of the sonotrode, since thevertical center of the sonicating area and the pivot point representedby the x-axis are mounted on substantially the same height. As a result,the upper portion of the side wall will remain in physical contact withthe sonicating area, while the lower portion—previously not incontact—will be rotated towards the sonicating area and touch it,thereby optimizing the possible contact area between the tube wall andthe sonicating area. In this position, the “sonication position”, boththe rotational movement of the sample tube holder and the translationalmovement of the sonotrode have reached an arresting point, such that theoptimized contact area is maintained for and throughout the sonicationprocess, as long as the force applied from the sonotrode to the sampletube and thus the sample tube holder is kept constant. The sonicationposition may, however, also be reached without a rotational movement, incase the contact area is already optimal upon primary contact of thesonicating area with the side wall of the sample tube.

Terms

The term “sonication”, as used herein, means a process of converting anelectrical signal into a physical vibration—in this case ultrasound—thatcan be directed toward the biological sample. An ultrasonic electricgenerator creates a primary signal powering a transducer. The signalhas, in some embodiments, a frequency of from 20 kHz, 35 kHz, or 50 kHzto 75 kHz, 100 kHz, or 250 kHz. In some embodiments, the frequency isabout 20 kHz. Via intermediate elements, such as crystals that may bepiezoelectric crystals stacked within the sonotrode, this signal istransformed into mechanical vibration, which process can also bereferred to as conversion. The sonotrode may be a rod such as a metalrod, and it may for instance have a cylindrical or conical shape. Thefrequency of the current may be chosen to be the resonant frequency ofthe sonotrode such that the latter vibrates lengthwise with standingwaves at its resonant frequency. The vibration may be amplified, forinstance, by a booster, before transmitting it to a sample tube via atransmittal unit comprising the sonicating surface of the sonotrode. Therapid movement of the sonicating face creates ultrasound waves migratingthrough the biological sample in the sample tube, inducing pressurevariations that grow and collapse. The corresponding kinetic energy isused for processing the biological material in the sample, for instance,in order to lyse cells such as mycobacterial or other cells or virusesof clinical interest.

A “sonotrode” usually comprises a stack of piezoelectric transducersattached to a tapering metal rod. Other suitable materials and shapesare known to the person of skill in the art. The end of the rod isapplied to the working material. An alternating current oscillating atultrasonic frequency is applied by a separate power supply unit to thepiezoelectric transducers. The current causes them to expand andcontract.

“Biological target material” or “biological material”, in the sense ofthis disclosure, comprise all kinds of biological molecules, for exampleproteins or nucleic acids, but also other molecules occurring in natureor being derivatives or synthetic analogues or variants thereof.Furthermore, the term “biological material” comprises viruses andeukaryotic and prokaryotic cells. In some embodiments, the biologicaltarget material is nucleic acids such as DNA, RNA or PNA. The DNA canbe, for instance, viral DNA, genomic DNA or plasmid DNA. The biologicaltarget material can be native or modified. Native biological material isnot irreversibly altered as compared the respective naturally occurringbiological material, such as e.g. DNA or RNA isolated from organisms.Modified biological material comprises biotinylated molecules such asnucleic acids or proteins, or the like.

As used herein, the term “biological sample” refers to biologicalmaterial that may potentially contain an analyte of interest. Inembodiments where the “biological sample” is a “liquid biologicalsample”, the sample can be derived from any biological source, such as aphysiological specimen, including blood, saliva, ocular lens fluid,cerebrospinal fluid, sweat, urine, stool, semen, vaginal fluid, milk,ascites fluid, mucous, synovial fluid, peritoneal fluid, amniotic fluid,tissue, cultured cells, sputum, or the like. The test sample can bepretreated prior to use, such as preparing plasma from blood, dilutingviscous fluids or diluting in general, lysis or the like. Methods oftreatment can involve filtration, distillation, concentration,inactivation of interfering components, and the addition of reagents. Abiological sample may be used directly as obtained from the source orused following a pretreatment to modify the character of the sample. Insome embodiments, an initially solid or semi-solid biological materialis rendered liquid by dissolving or suspending it with a suitable liquidmedium. In some embodiments, the biological sample is suspected tocontain a certain antigen or nucleic acid.

A “sample tube” means a vessel configured to hold the biological sample.A sample tube generally has a top and a bottom portion, wherein thebottom portion is typically closed while the top portion comprises anopening for introducing or removing the sample, reagents, or the like.The opening may be sealable, for instance, by a lid which may beremovable. Suitable lids are known to the skilled person and includescrew caps, form- or press-fit caps, sealing foils, and the like. Asample tube, as used herein, has a generally elongate shape, such as acylindrical, conical, or similar shape. It comprises a bottom wall and aside wall. The latter may be a continuous side wall or multiple sidewalls separated by angles. The inside surface of a sample tube isusually inert such that it does not interfere, for instance, withanalytical or preparative chemical reactions taking place within thetube. Sample tubes used in the devices and methods disclosed hereinusually have a length of from 2 cm, 3.5 cm, or 5 cm to 10 cm, 15 cm or20 cm. In some embodiments, the length from bottom to top is about 7.5cm. The width or diameter (side wall to side wall) is, in someembodiments, from 0.5 cm, 1 cm or 1.5 cm to 2 cm, 3.5 cm, 5 cm, in amore specific embodiment it is about 1 cm.

The “sample tube holder” refers to a device for receiving a sample tube.A sample tube holder can be a single-tube holder or a multiple-tubeholder. Multiple-tube holders include, for instance, racks for 2, 3, 4,5, 10, or more tubes. In some embodiments, the sample tube holder has aslot for inserting the sample tube and holding it in a specificposition. In the context described herein, the sample tube holderessentially fixes the sample tube, when inserted, in a specificorientation. In the absence of a rotational deflection of the sampletube holder, the sample tube holder holds the sample tube in anessentially vertical position. In some embodiments, the sample tube isform-fit within the sample tube holder, thus providing a tight fit in afixed orientation.

“Pivotally suspended”, in the context described herein, means that anobject such as the removable sample tube holder is engaged to anotherobject such as the corresponding mount with a rotational degree offreedom. The sample tube holder described herein can be rotated aboutthe x-axis as a pivot point while it is suspended from the mount.

The sample tube holder described herein is in some embodimentsremovable, wherein “removable” means that it can be separated from itscorresponding mount without any of the two components being destroyed.For instance, the sample tube holder may comprise a hinge that can beflexibly engaged to a corresponding bearing comprised by the respectivemount while allowing for a rotational degree of freedom. For example,after sonication of the sample in question, the sample tube holder maybe removed from the mount and the sonication device by disengaging thehinge of the sample tube holder from the corresponding bearing of themount. The sample tube holder may thus be transferred, for instance, toa biochemical analyzer or analytical module. Also, the sample tubes maybe filled, prepared or otherwise processed outside of the sonicationdevice while already held by the sample tube holder, thus bothsimplifying the workflow and reducing the risk of contamination due to,for example, spilling of sample or reagents within the sonicationdevice.

In some embodiments, the mount for the sample tube holder comprises twoparts, in some embodiments even more than two. In a more specificembodiment, the mount for the sample tube holder comprises an immobilebase portion and a rotatable portion comprising the pivot and a base forholding the sample tube holder, wherein the pivot is a hinge pivotallysuspended within a corresponding pivot bearing of the immobile baseportion.

The advantages of such a two-parted arrangement of the mount includethat, for instance, if the rotatable portion shows signs of abrasion,then this portion could be removed and replaced without having to affectthe immobile base portion. The latter can thus be permanently fixed tothe bottom of the sonication device as an integral part thereof.

In order to optimize alignment of the sonicating area of the sonotrodewith the biological sample contained in the sample tube when present inthe sample tube holder in its corresponding mount, the mount of thesonication device disclosed herein comprises in some embodiments aheight adjustment for adjusting the position of the sample tube in thesample tube holder on the z-axis. In this manner, a biological sample,particularly a liquid or partially liquid sample contained mostly in thebottom portion of the tube due to the effect of gravity, can be broughtto the height of the sonicating area of the sonotrode in the sonicationposition. This may avoid situations in which the sonicating area touchesa part of the tube wall where no or only little sample is located, whichmay render sonication of the sample more difficult. Means for adjustingthe height of the sample tube within the sample tube holder include, forinstance, a spring, a coil, a screw mechanism, an air buffer, anactuator, or the like. In some embodiments, the height adjustment ismanually operable. In other embodiments, the height adjustment comprisesan automated mechanism.

The “guiding rail” of the sonication device described herein can beembodied by any suitable structure providing guidance to the movement ofthe sonotrode in a defined orientation. In the simplest case, theguiding rail is a straight (one-dimensional) structure along which thesonotrode is moved back and forth by the actuator. However, the guidingrail may also extend across a second or third dimension whereappropriate. For instance, two or more sample tube holders mounted nextto each other may benefit from a guiding rail having a branching point,such that the sonotrode may be guided to each of the sample tube holdersin their respective mounts individually, as the application may require.The guiding rail may in some embodiments comprise a frame in which thesonotrode is suspended. Also in some embodiments, the guiding rail maybe embodied by a tunnel holding the sonotrode. In some embodiments, theguiding rail of the sonication device described herein comprises a sled.In a more specific embodiment, the sled is mounted on a railing andcarries the sonotrode on its top portion.

The sonication device described herein may comprise a control unitand/or a data management unit.

A “control unit” controls an automated system in a way that thenecessary steps for the processing protocols are conducted by theautomated system. That means the control unit may, for example, instructthe automated system to conduct certain pipetting steps with a pipettorto mix the liquid biological sample with reagents, or the control unitcontrols the automated system to incubate the biological sample orreagents or mixtures of both for a certain time at a certaintemperature, or the control unit controls the precise movements of thesonotrode of the sonication device described herein, or other movementsor related parameters. The control unit may receive information from adata management unit (DMU) regarding which steps need to be performedwith a certain sample. In some embodiments, the control unit might beintegral with the data management unit or may be embodied by a commonhardware. The control unit may, for instance, be embodied as aprogrammable logic controller running a computer-readable programprovided with instructions to perform operations in accordance with aprocess operation plan. In particular, the control unit may include ascheduler, for executing a sequence of steps such as the movementsdescribed above within a predefined time. The control unit may furtherdetermine the order of samples to be processed according to the assaytype, urgency, and the like. The control unit may also receive data froma detection unit related to a measurement of a parameter of the sample.

A “data management unit” is a computing unit for storing and managingdata. This may involve data relating to the liquid sample to beprocessed by the automated system, or data relating to the steps to becarried out by the sonication device described herein. The datamanagement unit may be connected to an LIS (laboratory informationsystem) and/or an HIS (hospital information system). The data managementunit (DMU) can be a unit within or co-located with the automated systemit interacts with. It may be part of the control unit. Alternatively,the DMU may be a unit remotely located from the automated system. Forinstance, it may be embodied in a computer connected via a network tothe automated system.

In order for the sonotrode to initiate a defined rotation of the sampletube holder about the x-axis, it may be advantageous to control and—ifrequired—adapt the force that is exerted from the actuator to thesonotrode and thus the sample tube in its corresponding holder.

Therefore, in some embodiments of the sonication device describedherein, the actuator is configured to apply a variable force to thesonotrode. In some embodiments, the actuator is configured to apply aforce of 10 N, 50 N, 100 N, or 500 N, to 750 N, 1000 N, 2500 N, or 5000N to the sonotrode. In a more specific embodiment, the force is about800 N.

Controlling and varying this force may be of advantage, for instance, ifdifferent types of sample tube holders are to be processed in the samesonication device. Based on, for example, the mass of the sample tubeholder, it may require an increased force to initiate the rotationalmovement about the x-axis, and the reset force may be greater than inthe case of a lighter sample tube holder. Furthermore, different typesof sample tubes may be used, wherein certain types may have a moredelicate structure than others. Hence, it may be useful to reduce theapplied force in order not to compromise the integrity of the morepressure-sensitive tube species.

To the benefit of parallelization and increased throughput, thesonication device described herein may be configured to support andprocess more than one sample tube at a time. This may require certainadaptations of the sample tube holder and/or the sonotrode, some ofwhich are disclosed herein as exemplary embodiments.

Thus, in some embodiments of the sonication device described herein, thesample tube holder is configured to hold multiple sample tubes and thesonicating area comprises one or more sub-areas with a concave surface.In some embodiments, the sonicating area comprises the same number ofsub-areas with a concave surface as the number of sample tubes that thesample tube holder is configured to hold.

A further aspect of the present disclosure is a method for sonicating abiological sample contained in a sample tube with an essentiallyround-shaped side wall using the sonication device disclosed herein, themethod comprising the following steps:

-   -   a) Inserting the sample tube into the sample tube holder, and        inserting the sample tube holder into the corresponding mount of        the sonicating device, wherein the order of these inserting        steps is interchangeable;    -   b) Moving the sonotrode along the y-axis and the guiding rail        towards the sample tube holder via the actuator until the        sonicating area contacts the side wall of the sample tube        through the opening;    -   c) Applying a pre-defined force from the actuator via the        sonotrode to the sample tube and thus the sample tube holder        along the y-axis such that the sonicating area contacts the        sample tube side wall and pushes the sample tube against the        inner wall of the sample tube holder or a rotatable portion        thereof, thereby firmly holding the sample tube in place and        adjusting its position along the x-axis by centering them with        the concave surface comprised by the sonicating area, the        movement of the sonotrode further causing a rotational movement        of the sample tube holder about the x-axis in case the side wall        is not aligned with the surface of the sonicating area until        reaching an arresting point, resulting in an enlarged contact        area between the sonicating area and the sample tube side wall;    -   d) Applying ultrasound to the sample tube from the sonotrode        through the contact area between the sonicating area and the        sample tube side wall.

Regarding step a), it may in some embodiments be of advantage to preparethe sample tube in the sample tube holder outside the sonication device,as described previously. For instance, multiple sample tubes may beplaced into a multi-tube sample tube holder and pre-processed, forexample, manually by laboratory personnel. Such a step could involveaddition of a liquid biological sample, along with or without a reagent,to the multiple sample tubes and capping the latter. In the next step,the laboratory personnel may conveniently place all the pre-processedsample tubes within the same sample tube holder into the sonicationdevice by engaging the sample tube holder to the corresponding mount.This facilitates the overall workflow and may lead to a reducedturnaround time. Alternatively, the sample tube holder may be placedinto the sonication device first. As an example, one of the multipletubes described before needs to be replaced, or an additional tube needsto be added, then the sample tube holder may be left inside thesonication device with the other tubes to be sonicated, while theindividual tube can be replaced or added.

As in the case of the sonication device described herein, it may beadvantageous to adjust the pre-defined force in step c) depending on thetype of sample tube and/or sample tube holder.

In some embodiments of the method described herein, step a) furthercomprises adjusting the height of the sample tube within the sample tubeholder.

Also in some embodiments of the method described herein, the sample tubeholder is configured to hold multiple sample tubes and the sonicatingarea comprises one or more sub-areas with a concave surface. Theadvantages of such embodiments include the increase of throughput, asdescribed in connection with the sonication device disclosed herein.

As set out above, vertical alignment of the sample in the sample tube onthe one hand and the sonicating area on the other hand can beadvantageous with regard to efficiency of the sonication of thebiological material in the sample. The same accounts for the lateralalignment by virtue of the concave surface of the sonicating area or itssub-areas, respectively.

All other specific embodiments of the method disclosed herein also applyto the device disclosed herein.

Exemplary Embodiments

The following Examples are meant to illustrate specific embodiments ofthe method and device disclosed herein, while they are not limiting.

The schematic drawing of FIG. 1A depicts a perspective side view of anembodiment of the sonication device (10) disclosed herein. In thisembodiment, the sample tube or tubes (300) and the sample tube holder(100) are omitted for the sake of better visibility of the othercomponents. Among the shown components is the corresponding mount (110)for the sample tube holder. The mount (110) in this embodiment comprisesan immobile base portion (160) and a rotatable portion (170) comprisinga base (171) for holding, in this case, a sample tube holder (100) withfive individual sample tubes (300). The rotatable portion (170) alsocomprises at both sides a pivot (130) whose pivot point defines thex-axis (150) about which the rotatable portion (170) and thus the sampletube holder (100), when inserted, is rotatable. The pivot (130), heredepicted as a hinge, is held by a corresponding pivot bearing (161) ofthe immobile base portion (160) of the mount. The rotatable portion(170) of this embodiment further has a height adjustment (120), in thedepicted case a manually operable mechanism based on screws (120) forlifting or lowering the base (171) holding the sample tube holder (100).In this embodiment, the entire sample holder (100) can thus bevertically adjusted, such that the sample tubes (300) and the biologicalsamples contained therein can be adjusted to the height of thesonicating area (210) of the sonotrode (200). Turning to the latter, itcan be seen that the sonication area (210) of this embodiment comprisesfive sub-areas (211) for the maximum load of five individual sampletubes (300). This sonicating area (210) of the sonotrode (200) facestowards an opening (140) to one side of the sample tube holder (100, notshown) such that it can be brought into physical contact with the sampletube or tubes (300) through the opening (140). The guiding rail (220) ofthe sonotrode (200) of the present embodiment comprises a sled (221) onwhich the sonotrode (200) is mounted, with the sled (221) itself beingmounted via a connector (222) to a railing (223). This guiding rail(220) arrangement provides movability of the sonotrode (200) along they-axis (250). It can be seen that this movability is one-dimensional,such that the sonotrode (200) can be moved with the help of an actuator(400) towards or away from the mount (110) for the sample tube holder(100), while the guiding rail (220) is fixed at one end to said mount(110), in this embodiment to its immobile base portion (160). The mount(110) is essentially in alignment with the z-axis (350), essentiallyparallel to the direction of gravity. Also visible in this depiction isthe composition of the sonotrode (200) of the embodiment shown here. Thepiezoelectric elements are contained within the converter (201)transforming a primary electrical signal into mechanical vibration. Thelatter is amplified by the subsequent booster (202), before thefollowing transmittal unit (203) transmits the energy via its sonicatingarea (210).

FIG. 1B shows the sonication device (10) of FIG. 1A in a sonicationposition. The sonotrode has been moved towards the sample tube holder(100) mounted to the rotatable part (170) of the corresponding mount(110). The sonicating area (210) with its individual sub-areas (211) arein contact with the sample tubes (300) held within the sample tubeholder (100) through the corresponding openings (140).

Turning to FIG. 2A, the sonication device (10) of FIG. 1 is now depictedas a cross-sectional side view. The sonicating area (210) is mounted atessentially the same height as the pivot (130).

Also visible is the vertical dimension of the sonicating area (210),along with its vertical center (215) that is mounted at the same heightas the pivot point defined by the x-axis (150).

As in FIG. 1B, a depiction of the sonication device (10) from thecross-sectional side-perspective of FIG. 2A is shown in a sonicationposition in FIG. 2B.

FIG. 3 and FIG. 4 provide an illustration of an embodiment of the methoddescribed herein using the sonication device (10) described herein asdepicted in the foregoing Figures.

FIG. 3A shows a perspective side view of the above-depicted embodimentof the sonication device (10) described herein. The guiding rail (220)has been omitted for the sake of better visibility of the mount (110)with the inserted sample tube holder (100). As in FIGS. 1A and 2B, thesonotrode (200) is located in a remote position, in which there is nodirect physical contact between the sonicating area (210) and the sidewall (310) of the sample tube (300). In contrast to the foregoingFigures, the rotatable portion (170) of the mount (110) is slightlytilted against the z-axis (350), which can be seen in more detail in thecorresponding cross-sectional side view of FIG. 4A.

The configuration of the sonication device (10) of this embodiment in asonication position is shown in FIG. 3B and FIG. 4B. Upon contact of thesonicating area (210) of the sonotrode (200) with the side wall (310) ofthe sample tubes (300) while applying a pre-defined force, the portionof the side wall (310) located above the vertical center (215) of thesonicating area (210) and thus the x-axis (150) being the pivot point ofthe pivot (130) is rotated counter-clockwise about the x-axis (150). Asthe sample tubes (300) are tightly fitted inside the sample tube holder(100), and the sample tube holder inside the rotatable part (170) of thecorresponding mount (110), the rotatable part (170) follows thisrotational movement of the sample tubes (300) until reaching thearresting point seen in present FIGS. 3B and 4B. In the resultingsonication position of this embodiment, the sample tubes (300) arevertical and thus parallel to the z-axis (350). This is due to thecircumstance that the side walls (310) of the sample tubes (300) shownin FIGS. 3 and 4 do not exhibit any substantial unevenness, and thesonicating area (210) of the sonotrode (200), including the sonicatingsub-areas (211) are aligned parallel to the z-axis (350). Hence, thevertical position of the sample tube (300) in the sample tube holder(100) and the rotatable part (170) of the mount (110) represents theposition in which the sample tube side wall (310) and the sonicatingarea (210) possess the optimal contact area, thus facilitating efficienttransfer of ultrasonic (kinetic) energy from the sonotrode (200) to thebiological sample. In the embodiment shown here, this means that each ofthe individual sub-areas (211) is in contact with the side wall (310) ofeach corresponding sample tube (300).

The drawings of FIGS. 5A-C provide further insight to the mechanism ofadjusting the sample tube (300) along the x-axis (250), displaying thesample tube holder (100)—more precisely, in the depicted embodiment, therotatable portion (170) of it—from various perspectives.

FIG. 5A depicts the movement of the transmittal unit (203) of thesonotrode (200) with its sonicating area (210) towards the sample tubeholder (100) in a chronological order. The rotatable portion (170) ofthe sample tube holder (100) is viewed from above as a cross-section atthe height defined by plane “A” as seen in FIG. 5B.

The first depicted stage of the movement is shown in FIG. 5A(i). Thesonicating area (210), in this embodiment comprising five distinctsonicating sub-areas (211), is already located at a short distance tothe side walls (310) of the sample tubes (300) held in the rotatableportion (170) of the sample tube holder (100). It can be seen that mostof the sample tubes (300) are not aligned with their correspondingsonicating sub-areas (211) along the x-axis (150). Turning to FIG.5A(ii), the sonicating sub-areas (211) come into physical contact withthe side walls (310) of the corresponding sample tubes (300). By virtueof their concave surface, the sonicating sub-areas (211) confer amovement of the sample tubes (300) along the x-axis (150) towards thecenter of each corresponding sub-area (211), which has progressedfurther in the depiction of FIG. 5A(iii). Through continued applicationof the pre-defined force moving the sonotrode (200) with its transmittalunit (203) towards the sample tube holder (100) along the y-axis (250),the concave sonicating sub-areas (211) effectuate a sliding movement ofthe sample tubes (300) with their essentially round-shaped side walls(310). It can be seen that the embodiment shown here involves supportingelements (171) protruding from the inner back wall of the rotatableportion (170) of the sample tube holder (100). These protrusions exhibita concave surface in analogy to the surface of the sonicating sub-areas(211) and contribute to aligning the sample tubes (300) in x-direction.However, the concave surfaces of the sonicating sub-areas (211) arecapable of bringing about the alignment also by pressing the sampletubes (300) against a flat surface of the back wall. Hence, in someembodiments, the back wall does not comprise the supporting elements(171) shown here. An end point is reached in FIG. 5A(iv), where thesample tubes (300) are aligned along the x-axis (150) with theircorresponding sonicating sub-areas (211). The person skilled in the artwill appreciate that the sonicating area (210) need not necessarily beembodied, as depicted here, as a surface supporting individualsonicating sub-areas (211). For instance, the sonicating area (210) mayhave a single concave surface without any protruding sub-areas (211)which may, for example, be arranged for engaging a single sample tube(300). In some embodiments, the rotatable part (170) of the mount (110)may be flexibly suspended such that one or more degrees of freedom aregiven in which the rotatable part (170) may contribute to optimalalignment of the components involved. For example, the pivot can slidewithin the pivot bearing (161) of the immobile base portion (160) of themount along the x-axis to provide additional lateral movement of thetube or tubes to optimize contact between the sonotrode sonicatingsub-areas and the sample tube or tubes.

1. A sonication device for sonicating a biological sample contained in asample tube with an essentially round-shaped side wall, the sonicationdevice comprising: a. a sample tube holder pivotally suspended in acorresponding mount, allowing for a rotational movement of the sampletube holder in vertical direction about an x-axis being a rotationalaxis of a pivot, wherein the sample tube holder is configured, in theabsence of a rotational movement, to hold the sample tube in a verticalorientation along a z-axis being essentially parallel to the directionof gravity and perpendicular to the x-axis, and wherein the sample tubeholder comprises an opening towards at least one side; b. a sonotrodecomprising a sonicating area for applying ultrasound to the sample tube,the sonicating area comprising a concave surface and being mounted atessentially the same height as the pivot with the x-axis beingessentially at the height of the vertical center of the sonicating area,wherein the sonotrode is mounted on a guiding rail for moving thesonotrode along a y-axis perpendicular to the x-axis and to the z-axistowards or away from the sample tube holder, wherein the mount for thesample tube holder is fixed to one end of the guiding rail with theopening of the sample tube holder facing the sonicating area such thatthe sonicating area in a sonication position is in physical contact witha side wall of the sample tube in the sample tube holder; and, c. anactuator for moving the sonotrode on the guiding rail along the y-axisrelative to the sample tube holder.
 2. The sonication device of claim 1,wherein the sample tube holder is removable.
 3. The sonication device ofclaim 1, wherein the mount for the sample tube holder comprises animmobile base portion and a rotatable portion comprising the pivot and abase for holding the sample tube holder, wherein the pivot is a hingepivotally suspended within a corresponding pivot bearing of the immobilebase portion.
 4. The sonication device of claim 1, wherein the mount forthe sample tube holder further comprises a height adjustment foradjusting the position of the sample tube in the sample tube holder onthe z-axis.
 5. The sonication device of claim 1, wherein the guidingrail comprises a sled.
 6. The sonication device of claim 1, wherein theactuator is configured to apply a variable force to the sonotrode. 7.The sonication device of claim 6, wherein the actuator is configured toapply a force of 10 N, 50 N, 100 N, or 500 N, to 750 N, 1000 N, 2500 N,or 5000 N to the sonotrode.
 8. The sonication device of claim 7, whereinthe force is about 800 N.
 9. The sonication device of claim 1, whereinthe sample tube holder is configured to hold multiple sample tubes andthe sonicating area comprises one or more sub-areas with a concavesurface.
 10. The sonication device of claim 1, wherein the correspondingmount is configured to permit movement of the sample tube holder alongthe x-axis.
 11. The sonication device of claim 1, wherein the sampletube holder is configured to permit movement of the sample tube withinthe sample tube holder along the x-axis.
 12. A method for sonicating abiological sample contained in a sample tube with an essentiallyround-shaped side wall using the sonication device of claim 1, themethod comprising the following steps: a) inserting the sample tube intothe sample tube holder, and inserting the sample tube holder into thecorresponding mount of the sonicating device, wherein the order of theseinserting steps is interchangeable; b) moving the sonotrode along they-axis and the guiding rail towards the sample tube holder via theactuator until the sonicating area contacts the side wall of the sampletube through the opening; c) applying a pre-defined force from theactuator via the sonotrode to the sample tube and thus the sample tubeholder along the y-axis such that the sonicating area contacts thesample tube side wall and pushes the sample tube against the inner wallof the rotatable portion of the sample tube holder, thereby firmlyholding the sample tube in place and adjusting its position along thex-axis by centering them with the concave surface comprised by thesonicating area, the movement of the sonotrode further causing arotational movement of the sample tube holder about the x-axis in casethe side wall is not aligned with the surface of the sonicating areauntil reaching an arresting point, resulting in an enlarged contact areabetween the sonicating area and the sample tube side wall; and, d)applying ultrasound to the sample tube from the sonotrode through thecontact area between the sonicating area and the sample tube side wall.13. The method of claim 12, wherein the sample tube holder is configuredto hold multiple sample tubes and the sonicating area comprises one ormore sub-areas with a concave surface.